Working canines are often deployed to contaminated environments with unknown chemical and biological content. Little data exists supporting the effectiveness of current canine decontamination protocols. The objective of this study was to prospectively compare the effectiveness of a standard decontamination protocol utilized in real-world working scenarios by Federal Emergency Management Agency (FEMA) urban search and rescue teams (Protocol A) vs. a modified protocol (Protocol B) developed from technician feedback. Protocol A (n = 7) utilized stiff bristle brushes, generic pet shampoo, and a double rinse system. Protocol B (n = 7) utilized a soft rubber grooming brush, Johnson & Johnson's ® Head-to-Toe Body Wash, and a grated floor in addition to the existing rinse system to facilitate drainage of gray water. An oil-based pseudo-contaminant (Glo-Germ) was topically applied to four anatomical sites on each canine: the throat latch; between the shoulder blades; the inner aspect of the hind leg; and the hind paw. Reduction in contamination was assessed after each canine underwent decontamination and scored using a previously validated scale. Categorical data were analyzed using a Chi Square test and PROC FREQ of SAS (version 9.4). Overall, Protocol B was associated with greater reduction in contamination compared to Protocol A (P = 0.01), with the throat latch and inner hind leg more likely to retain contaminant than the hind paw or between the shoulder blades (P < 0.01). Canines decontaminated using Protocol A were more likely to have residual contamination of the throat latch or inner hind leg compared to the hind paw (P < 0.05). In contrast, those decontaminated using Protocol B were likely to have residual contamination of only the throat latch compared to other sites (P < 0.05). Simple modifications in current FEMA protocols increased the overall effectiveness of working canine decontamination.