Microbial enrichments with a substrate of interest could enhance the possibility of finding certain desired metabolic activities. As lipases are one of the most important enzymes in industrial applications, the Colombian Andean soils were explored as a source of lipolytic microorganisms. Two Andean soils under low temperatures were sampled: paramo and glacier soils from “Los Nevados” National Natural Park. Both soils were enriched through a fed-batch fermentation using olive oil as the inductor substrate. Forty-three lipolytic isolates were obtained and their taxonomic assignments were performed on the basis of 16S rDNA gene sequencing. In both cases, the phylum Proteobacteria represented the majority of the isolates. Qualitative assays to measure the lipolytic activity were performed by using tributyrin, triolein or olive oil (1%). Two isolates identified as Pseudomonas psychrophila and Stenotrophomonas rhizophila produced the largest hydrolysis halos, with an optimal activity at pH 8 and 50°C. Only the S. rhizophila extracellular fraction hydrolyzed short and long chain pNP-esters, including pNP-palmitate. The broader substrate specificity of this isolate is probably due to the simultaneous presence of lipase and esterase activity in the crude extract. This is the first report of lipolytic activity in S. rhizophila, and the first preliminary characterization of the novel lipolytic activity in S. rhizophila and P. psychrophila. Further work is needed to purify and completely characterize the esterases and lipases produced by both species.