Objective To compare and analyze the germicidal efficacy of continuous ultraviolet and pulsed ultraviolet (UV) lasers on pathogenic microorganisms. Methods Spore slides of Escherichia coli and Bacillus atrophaeus were irradiated using a 254 nm ultraviolet mercury lamp and UV laser. The carrier quantitative germicidal test was conducted to determine the disinfection efficacy at different irradiation doses. Results When the irradiation dose of the ultraviolet mercury lamp was 245.52 mJ/cm², the logarithmic killing values of Escherichia coli and Bacillus atrophaeus spores were 5.00 and 2.92, respectively, and the mean logarithmic killing doses were 49.10 and 84.08 mJ/cm², respectively; When the UV laser irradiation doses were 208.39 and 206.80 mJ/cm², the logarithmic killing values for the two microorganisms were 6.29 and 3.32, respectively, and the mean logarithmic killing doses were 33.13 and 62.29 mJ/cm², respectively. Conclusion Compared with continuous UV radiation, pulsed UV laser has stronger penetration ability, better killing efficacy on pathogenic microorganisms at the same radiation dose, and can conduct targeted disinfection and sterilization on the surface of objects directionally.