Removal of hexane from air–hexane mixtures in biofilters packed with different solid media under nitrogen supplementation was performed for 70 days. Two columns containing Perlite or a mixture of peat and Perlite, were used. The solid media were supplemented with nitrogen source up to 1 kg/m3 per week for high nutrient supplementation and 0.2 kg/m3 per month for low nutrient supplementation. A high rate of hexane removal: 95 g/m3 h was achieved under high nutrient supplementation, high air flow rate and high hexane concentration. However, the percentage of hexane removal decreased with increasing air flow rate and hexane inlet concentration. For high nutrient supplementation the type of solid medium did not significantly affect the biodegradation capacity. With low nutrient supplementation, the highest removal rate was achieved in the column containing the peat–perlite mixture. The column containing perlite had a significantly lower pressure drop (20 Pa/m) than the 2400–2930 Pa/m observed for the column containing the mixture. Perlite offers an opportunity of running a biofiltration process at a lower and stable pressure drop if the nutrient supplementation is managed properly.