Aim: Multidose vials (MDVs) for local anesthetic injections are routinely used in dental practice. MDVs contain multiple doses of a parenteral drug intended for administration to the same or multiple patients. Potentially harmful microbes have been shown to be able to live and occasionally multiply in MDVs if not handled aseptically. The goal of this study thus was to evaluate the bacterial and fungal contamination (FC) of lignocaine MDVs after use in a dental hospital. Materials and methods: A total of 27 MDVs of lignocaine free of any microbial contamination were distributed to different departments of the dental hospital and they were asked to use them on patients routinely. The samples were recollected from the departments either at the end of the 28th day or as and when the contents in the MDVs reached a predetermined level marked on the bottle during its usage, whichever was earlier. These leftover samples were subjected to a microbiological investigation by inoculation into thioglycolate broth and subsequent subculturing onto agar plates. Results: None of the inoculated broths showed turbidity. Subcultured agar plates, even on the 7th day of incubation, did not show any bacterial or fungal growth. The lignocaine MDVs tested after use were thus found to be sterile. Conclusion: There was no bacterial or FC detected in MDVs of lignocaine subjected to testing after use in the dental hospital. Clinical significance: Multidose vials (MDVs) continue to be used in clinics for economic reasons. But the clinician opting for MDVs should be conscious of their potential for microbial contamination and should meticulously follow the protocol for their aseptic use.