Dental practitioners should be able to detect and identify various oral lesions and hence provide an accurate diagnosis and treatment plan to the patient for a better prognosis. Some oral lesions that give similar clinical appearances must be confirmed by adjunctive procedures such as biopsy. They should be knowledgeable about various techniques used for oral biopsy and should have the ability to understand and formulate a treatment plan based on the biopsy report. Thus, the aim of the present study was to carry out a questionnaire-based survey of dental practitioners in Madurai to assess their knowledge, practice, and attitude regarding biopsy procedures. This cross-sectional questionnaire-based study was done on 180 dental practitioners in Madurai, consisting of 17 questions formulated to collect information through an online platform on various aspects of oral biopsy. The data thus collected was tabulated. The response rate was 90%. Most of the DPs had a good knowledge about biopsy, but there were conflicting results on performing biopsy. Most of the dental surgeons are not comfortable performing biopsies; the majority seem to lack experience and skill. There is a need to organize training for dental surgeons to enhance their practical skills.