Mycoplasma contamination in cell culture is a serious setback to cell culturists across the world with a very high rate of reported occurrence particularly because of difficult early detection. Out of a variety of detection methods known, the double-step nested polymerase chain reaction (PCR)-based detection of mycoplasma in cell culture has been critically viewed upon because of chances of producing reliable results. A nested PCR technique, described to detect a large range of cell-culture-contaminating mycoplasma species, with greater sensitivity to detect as low a contamination as a few organisms, was compared with the results from two cytological techniques employed in tandem. These are DNA staining using Hoechst, the gold standard, and an immunofluorescent assay using a highly specific monoclonal antibody. The study undertaken on randomly collected cell cultures revealed a false-negative and several false-positive results in comparison to the cytological methods employed. The observations were particularly more unambiguous with the immunofluorescent assay employed in the study while simultaneously employed Hoechst staining serving as an indicator of bacterial contamination. There is a general apprehension that genus-specific PCR approaches could be associated with inaccurate outcome and only species-specific PCRs may be satisfactory in routine screening for mycoplasma contamination in cell cultures. At this juncture, it may be suggested that caution must be exercised while adopting the two-step nested PCR-based detection approaches, and the simultaneous employment of cytological methods used in this investigation could prove to be practicable in the proper interpretation of results.