Aims: The performance of three scanning CO2 laser inactivation systems was assessed and included: a gantry system, a rapidly rotating mirror and a low-power hybrid system combining an oscillating mirror and rotary motion of the sample. Methods and Results: Escherichia coli and Staphylococcus aureus were the target organisms on stainless steel, nutrient agar or moist collagen film and the laser power was varied from 2 to 1060 W (two laser sources). In general, a threshold energy density was identified, above which no inactivation was observed because the scanning velocity was too high (10 cm s(-1) for stainless steel, 660 W). Reducing the velocity increased the inactivation process until complete inactivation was observed at 1.3 cm s(-1) (E. coli, similar to 10(6) CFU per sample) and 0.82 cm s(-1) (S. aureus, similar to 10(8) CFU per sample); consequently, S. aureus organisms showed a greater resistance to laser irradiation. For the nutrient agar and collagen samples, the averages of the width of clearing were measured as a function of the translation velocity and the rates of inactivation (I-R, cm(2) s(-1)) were found; an optimum velocity was observed that produced the maximum rate of inactivation. At a laser power of 1060 W, the maximum value of I-R was 140 cm(2) s(-1) (similar to 10(7) CFU cm(-2)) for S. aureus on collagen and slightly less on nutrient agar (114 cm(2) s(-1), estimated from a best-fit polynomial, r(2) = 0.98). Conclusions: A comparison of the low- and high-power lasers produced values of 0.09 cm(2) s(-1) W-1 (i.e. I-R per Watt delivered) for S. aureus on nutrient agar with the low-power laser at 13 W and on collagen 0.13 cm(2) s(-1) W-1 for 1060 W. The rate of inactivation was found to be a function of the laser power, translation velocity and properties of the substrate media. The three laser inactivation systems successfully demonstrated the potential speed, efficiency and application of such systems. Significance and Impact of the Study: Laser scanning systems offer the potential for rapid and efficient inactivation of surfaces, eliminating the need for chemical treatment.