The present investigation was to study the effect of different non-ionic surfactants (Tween-80, Tween-60, Tween-40, Tween-20, Triton X-100) and a rhamnolipid biosurfactant on the degradation of fluorene by Paenibacillus sp. PRNK-6. An enhancement in the growth, as well as fluorene utilization by this strain were observed in the presence of biosurfactant and non-ionic surfactants except Tween-20 and Triton X-100. Triton X-100 and Tween-20 were toxic to this bacterium. The strain PRNK-6 utilized 75% of fluorene (280mg/L) in 24h in an unamended condition. On the other hand, the complete utilization of higher concentration fluorene (320mg/L) by this strain was noticed when the medium was amended with Tween-80 (1.5% v/v) within 24h of incubation. Whereas, 90.6%, 96.5% and 96.7% of fluorene (280mg/L) was utilized when amended with Tween-60 (3.5% v/v), Tween-40 (3% v/v) and biosurfactant (25mg/L) respectively. Biosurfactant promoted the fluorene degradation potential of PRNK-6 as 96.2% of 320mg/L fluorene was degraded within 24h. Further, the added tween series surfactants and a biosurfactant have increased the cell surface hydrophobicity of the PRNK-6. Thus correlating with the enhanced degradation of the fluorene.