This research aimed to characterize bio-surfactants produced by Bacillus subtilis 3KP, Pseudomonas putida T1-8, Micrococcus sp. L II 61 and Acinetobacter sp. P 2(1) and to investigate its combination's effects on the removal of crude oil in nonwoven fabric with different washing times vary from 12, 24 to 36 hours. The production of bio-surfactants was done on Synthetic Mineral Water mixed with molasses 4% within four days. The bio-surfactant products were characterized by measuring the Surface Tension (ST) (mN/m) and Emulsion Activity (EA) (%). Oil removal experiment was done by mixing 10 mL bio-surfactant with nonwoven fabric that contains crude oil into 50 mL bottle inside a shaker. The removed crude oil was extracted with n-hexane and measured gravimetrically. The results were then being analyzed with two ways ANOVA and Duncan test. Bio-surfactant produced by four bacteria has variations of Surface Tension and Emulsion Activity values. Bio-surfactant produced by Bacillus subtilis 3KP and Pseudomonas putida T1-8 showed the increasing of crude oil removal as washing times increase, while bio-surfactant produced by Micrococcus sp. L II 61 and Acinetobacter sp. P2(1) showed the decreasing result at 36 hours. However, the combination that showed the best result was Acinetobacter sp. P 2(1) at 24 hours valued 65,3%.