Comprehensive childhood obesity prevention (COP) strategies should include increasing school nurse involvement. This study was conducted to determine the influence of key school nurse perceptions (self-efficacy, perceived benefits, and perceived barriers) on participation in COP practices at the individual child and school level. Florida registered nurse (RN) school nurses (n = 171) anonymously completed online or paper questionnaires. Linear regression analyses identified a model of self-efficacy with perceived benefits and barriers that explained 12% and 9.1% (p < 0.001) of variance in child-level and school-level COP practices, respectively. Self-efficacy explained the most variance in both models (p < 0.001). Mediation testing identified perceived barriers as a partial mediator of the influence of self-efficacy on child-level practices. These findings support interventions and policy changes to increase self-efficacy and reduce perceived barriers to promote school nurse involvement in preventing childhood obesity.