The regulation of biofilm development requires multiple mechanisms and pathways, but it is not fully understood how these are integrated. Small RNA post-transcriptional regulators are a strong candidate as a regulatory mechanism of biofilm formation. More than 200 small RNAs in the P. aeruginosa genome have been characterized in the literature to date; however, little is known about their biological roles in the cell. Here we describe the identification of the novel regulatory small RNA, SrbA. This locus was up-regulated 45-fold in P. aeruginosa strain PA14 biofilm cultures. Loss of SrbA expression in a deletion strain resulted in a 66% reduction in biofilm mass. Furthermore, the mortality rate over 72 hours in C. elegans infections was reduced to 39% when infected with the srbA deletion strain compared to 78% mortality when infected with the parental wild-type P. aeruginosa strain. There was no significant effect on culture growth or adherence to surfaces with loss of SrbA expression. Also loss of SrbA expression had no effect on antibiotic resistance to ciprofloxacin, gentamicin, and tobramycin. We conclude that SrbA is important for biofilm formation and full pathogenicity of P. aeruginosa.