Introduction: Dental unit waterline system (DUWLS) decontamination is an important infection control in dental practice. This study aimed to determine knowledge and attitude towards DUWLS decontamination and its associated factors among undergraduate and postgraduate dental students in Universiti Sains Malaysia. Methods: A cross-sectional study was conducted using a validated questionnaire administered online using Google Forms to assess the knowledge and attitude of dental students towards DUWLS decontamination. Descriptive and inferential statistics using independent t-test, Pearson, and Spearman correlation were carried out using SPSS version 24.0. Results: A total of 169 dental students participated in the study, and the majority were female (71.6%) and Malay (59.2%). The mean (SD) age was 23.2 (0.93) and 34.4 (2.65), for the undergraduates and postgraduates, respectively. The mean (SD) knowledge scores for the undergraduates and postgraduates were 11.6 (4.23) and 18.0 (2.82), respectively, whereas the mean attitude scores were 51.3 (7.86) and 54.6 (4.74), respectively. The postgraduates had significantly higher mean knowledge and attitude scores than the undergraduates (p<0.001). The significant factors associated with the undergraduates’ knowledge were received information and guidance and their personal experience in managing DUWLS (p<0.05). However, only received information on DUWLS was significantly associated with the postgraduate’s knowledge score (p=0.011). Age factor had a significant association with the attitude score among the postgraduates (p=0.014). Conclusion: The knowledge regarding DUWLS decontamination was relatively moderate in both groups. However, both groups showed a favourable attitude towards DUWLS decontamination. Significantly higher knowledge and attitude scores were presented among the postgraduates than the undergraduates.