Background The accuracy of the TB diagnosis depends on the quality of the collected and transported samples. Inadequate knowledge and practices regarding the collection and transport of sputum samples can lead to false-negative results and delay the diagnosis and treatment of TB.This study was conducted to assess the knowledge of residents and interns about the collection and transport of sputum and other samples for the diagnosis of tuberculosis in a medical college hospital. Methods This was a cross-sectional study conducted among 120 medical interns and postgraduate residents of a medical college hospital in coastal Karnataka, India. Participants were interviewed using a pre-designed and structured questionnaire. The data was analyzed using IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. Descriptive statistics like the mean, standard deviation, and chi-square test were used. A p-value less than 0.05 was considered statistically significant. Results Most of the study participants (76, 63.3%) belonged to the age group of 22 to 25 years and were interns. Nearly three-quarters of the participants (85, 70.8%) were female. Based on the mean score, 69.2% of the participants exhibited good knowledge. Among the socio-demographic characteristics, being a postgraduate resident was associated with good knowledge about the collection and transport of samples (p-value< 0.05). Conclusion Seven out of ten participants had good knowledge about the collection and transport of sputum and other samples for the diagnosis of tuberculosis. Strengthening the training of this category of health workers needs to be prioritized.