Oral opportunistic pathogens from poor oral health can cause problems among elderly and immunocompromised individuals, such as lower respiratory tract infection or aspiration pneumonia (AP). Statistically, 11.4% of deaths in Malaysia were caused by pneumonia which has been categorized as one of the main causes of death. The main objective of this study is to evaluate the antibacterial effect of crude n-hexane Alpinia conchigera rhizome extract (ACRE) and its major bioactive compound, 1’S-1’-acetoxychavicol acetate (ACA) against selected oral opportunistic pathogens which consist of clinical and ATCC strains of Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae), Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa). In this study, a total of 13 respondents were recruited to obtain the clinical isolates of selected oral opportunistic pathogens. From these samples, 3 strains of S. aureus, 1 strain of S. pneumoniae, 3 strains of K. pneumoniae and 1 strain of P. aeruginosa were obtained and further tested. To achieve the objective, disc diffusion assay (DDA), minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill assay were carried out to determine the antibacterial properties while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were carried out to observe the morphological changes on the affected clinical strain of S. pneumoniae. To identify the effect of ACA on the gene expression of S. pneumoniae clinical strain, RT-qPCR were employed. Based on DDA results, ACRE-n-hexane and ACA displayed good antibacterial activity against clinical and ATCC strains of S. aureus, S. pneumoniae and P. aeruginosa with the zone of inhibition recorded at 25.07 ± 0.09 mm, 36.83 ± 0.85 and 14.00 ± 0.82 respectively while clinical and ATCC strains of K. pneumoniae did not show any inhibition diameter. The range of MIC and MBC values for ACRE-n-hexane recorded were between 3.13 mg/mL and 50.00 mg/mL while ACA between 0.39 mg/mL until 12.50 mg/mL. ACRE-n-hexane and ACA exhibited both bacteriostatic and bactericidal properties mostly when treated with concentration of 3 × MIC, 2 × MIC, MIC and ¾ MIC at different time intervals. The results from electron microscopy indicated that ACA showed the ability to cause cell wall disruption, decreasing cell membrane integrity and morphological disorder towards the clinical strain of S. pneumoniae. The virulence genes of psaA and pavA that functions as adherence factor for S. pneumoniae were suppressed while other genes such as cbpA, cbpD, nanA and pspA were expressed after treated with ACA at the concentration of 6.25 mg/mL. In conclusion, ACRE-n-hexane and ACA possess antibacterial effect against clinical and ATCC strains of S. aureus, S. pneumoniae, K. pneumoniae and P. aeruginosa. The effects of ACA are reflected in the morphological and ultrastructural changes and the gene expression of S. pneumoniae clinical strain.