: Pseudomonas aeruginosa a gram-negative bacterium with high versatility that can undergo aerobic and anaerobic respiration. Capabilities in deploying different carbon sources, energy metabolism, and regulatory system, ensure the survival of this microorganism in the diverse environmental conditions. Determination of differences in carbon sources utilization among biofilm and non-biofilm of P. aeruginosa provides a platform in understanding the metabolic activities of the microorganism. Three archived clinical isolates of strong, moderate, and non-biofilm producers were identified. ATCC 27853 P. aeruginosa was used as a negative control or non-biofilm producing microorganism. Biofilm formation of ATCC 27853 P. aeruginosa was confirmed by crystal violet assay (CVA) and Congo red agar (CRA). Metabolic profiles of the biofilm and non-biofilms isolates were determined by phenotype microarrays (Biolog Omnilog). In this study, the biofilm isolates utilized uridine, threonine, and serine while non-biofilm isolates utilized adenosine, inosine, monomethyl, sorbic acid, and succinamic acid. Sugar utilization was seen between the isolates adhering that biofilm P. aeruginosa preferred glucoside, galactoside, glucuronide, glucosaminide, mannoside, and galactosaminide, but it was not utilized by the non-biofilm. The outcome of this result can be used for future studies to improve detection or inhibition of the growth of clinical P. aeruginosa biofilm and non-biofilm, respectively.