Abstract: Background and Objective: Biofilm formation is important for the establishment of bacterial pathogenesis and disease control. Formation of cell aggregates has contributed to the long-term colonization of renal tubules of mammalian maintenance host by pathogenic Leptospira. This study aimed to quantify the biofilm formation among 29 pathogenic strains of Leptospira isolated from rats, soil and water samples in Sarawak, Malaysia. Materials and Methods: A starting bacterial suspension inoculum of about 106 bacteria mL–1 was prepared from mid-exponential cultures. Biofilm assay was then conducted in triplicate in 24-well microtitre plates. Crystal violet assay was performed to assess the biofilm forming abilities based on optical density obtained. Based on adherence strength, the biofilm forming abilities were classified into four different categories: Non-adherent, weakly adherent, moderately adherent and strongly adherent. Results: A 32.26% each of tested bacteria was classified either as non-adherent or weakly adherent biofilm producers on 1st day. From 2nd to 5th day, most of them produced moderately and strongly adherent biofilms. All the isolates adhered strongly from 6th to 10th day. Highest biofilm production was noticed either on 7th to 8th day. In this study, the strongest biofilm producers among rats, soil and water samples are P38 with OD600 at 16.700±0.265 on 8th day, P18 with OD600 at 21.760±0.332 on 7th day and P22 with OD600 at 19.793±0.144 on 7th day, respectively. Conclusion: In conclusion, all the tested Leptospira were able to produce strong biofilm, which contributed to the survival in diverse environmental habitats and anticipated in disease transmission.