Introduction. Non-contact condensing lenses (NCLL) are a requirement in ophthalmology examinations. To date, there have been no studies on the types of bacteria found on handheld lenses used to examine patients in an ophthalmology clinic. Hypothesis. The BD BACTEC Peds Plus broth (BACTEC-PP) culture method can isolate more organisms as compared to conventional culture plates (CCP) from NCLL. Aim. To evaluate the organism spectrum cultured from NCCL for fundus examination and to compare the results between BACTEC-PP and CCP. The isolation results were then related to the participant’s knowledge and hand hygiene practices (HHP). Methodology. This is a comparative cross-sectional study involving consenting trainee ophthalmologists from a single centre, whose preferred NCCL was swabbed from January to December 2019. The respondents completed the adapted World Health Organization Hand Hygiene Knowledge and Perception Questionnaire, and their HHP were observed by Infective Control Unit nurses. Positive bacterial growth using both methods, in addition to hand hygiene practices, were compared. Results. All samples had positive yields by at least one method. BACTEC-PP had a higher yield of 47 (90.4%) isolates compared to CCP with 37 (71.2%) isolates, P=0.041. CoNS sp (38.9 %) was the most common isolate with both methods, followed by Bacillus sp (25.3 %). At the same time, three fungi were detected with CCP only (3.2 %). There was a significant correlation in bacterial isolation with years of training, with fewer isolates among seniors in both BACTEC-PP (P=0.049) and CCP (P=0.034). There were no significant correlations between HHP and positive yields from either culture method. Conclusions. NCCL used by trainee ophthalmologists are typically contaminated by at least one bacteria, with CoNS sp being the most commonly isolated. More positive cultures occurred in lenses from junior trainees. Contamination was not correlated with knowledge or HHP. BACTEC-PP has significantly higher yields than CCP for bacterial isolation from NCCL, but did not isolate any fungus.