Background: The vaginal microbiota consists primarily of Lactobacillus species, which help maintain microbial balance through acid production and antimicrobial activity. The disruption of balance causes vaginal dysbiosis and infection. Objective: The aim of this study was to analyze the association of vaginal infection with the distribution of Lactobacillus species, vaginal pH, and antimicrobial activity. Methods: The study was designed as a case-control study. A total of 100 subjects, including 50 healthy subjects and 50 infected subjects, were included. Vaginal swabs were collected to assess pH, microbial isolation, and identification using the standard microbiology technique. The antimicrobial activity of Lactobacillus isolates was assessed using the agar well diffusion technique and peroxide production was assessed qualitatively. Chi-square test and independent t-test were used for the statistical analysis of the results. Results: The isolation of Lactobacillus was 90% in healthy women and 40% in infected women, with a statistically significant difference (p < 0.001). Infected women had increased vaginal pH (5.6 ± 0.5) as compared to healthy women (4.3 ± 0.3) (p < 0.001). Lactobacillus crispatus was the most frequent in healthy women and Lactobacillus iners was the most frequent in infected women. Pathogenic organisms, especially Gardnerella vaginalis, were statistically significantly more common in the infected group. The isolates taken from healthy women had the highest hydrogen peroxide production and antimicrobial activity. Conclusions: An increase in vaginal pH, a decrease in antimicrobial defenses, and overgrowth of pathogens are associated with the loss of functionally active Lactobacillus species. Composition and functional capacity are critical factors in determining vaginal health.