Purpose Health professionals, especially in dentistry, are involved at high risk of microbial cross contamination. There are many studies that reported the risk of cross-contamination in prosthetic laboratories. Although much importance has been given to infection control practices, such as the barrier technique, sterilization, and disinfection of the dental office and instruments, less emphasis has been given for the disinfection of dentures. The dentures in mouth are prosthetic medical devices that create an appropriate habitat for both pathogenic and nonpathogenic organisms to nurture. To avoid cross-contamination, denture disinfection is mandatory. This study evaluated the efficacy of four denture disinfectants on four most common organisms found in denture biofilm at two different time intervals. Materials and Methods Three hundred twenty denture base acrylic resin specimens were fabricated by means of wax patterns with dimensions diameter 15 mm × 4 mm. The contamination of samples was done by the four microorganisms taken in study that were Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, and Escherichia coli. The contaminated samples were disinfected by immersion procedure in four disinfectants that were sodium hypochlorite, glutaraldehyde, peracetic acid, and chloroxylenol. Results Sodium hypochlorite was the best disinfectant resulting in complete elimination of microorganisms in 5 minutes immersion time. Glutaraldehyde and chloroxylenol were also effective against microorganisms. Minimum 10 minutes immersion time was required to completely eliminate the microorganisms. Peracetic acid was least effective of all showing the least reduction in microbial growth. Among the organisms, S. aureus showed the maximum resistance to disinfection, and C. albicans was the least resistant.