The fatty acid composition of bacterial isolates remains stable under standardized culture conditions, which makes it a useful taxonomic marker. The present study aims to characterize the diversity and quantity of fatty acid methyl esters (FAME) profiles of cultivable bacterial isolates collected along the southwest coast of India. Based on the similarity indices (range > 0.3-0.7) of the FAME profiles, the isolates were aggregated into 10 families, 11 genera and 19 species of cultured isolates. The following classes of bacteria were found: Bacilli, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Actinobacteria, which also included a few pathogens such as Pseudomonas, Staphylococcus and Bacillus sp. The hydroxyl FAMEs 2-hydroxydodecanoic acid (C-12:0 2OH), 2-hydroxypentadecanoic acid (C-15:0 2OH),3-hydroxy 14-methylpentadecanoic acid (C(16:0)iso 3OH), 3 hydroxy hexadecenoic acid (C-16:0 3OH) and 3-hydroxy 15-methylhexadecanoic acid (C(17:0)iso 3OH), as well as the unsaturated FAMEs (11Z)-11-hexadecenoic acid (C-16:1 x277;5c), were exclusively associated with the isolates from Mangalore samples. Similarly, FAMEs 2-hydroxydecanoic acid (C-10:0 2OH), 9-methyldecanoic acid (C(11:0)iso), undecanoic acid (C-11:0), tridecanoic acid (C-13:0), 10-methylhexadecanoic acid (C-16:0 10-CH3) and (7Z)-7-hexadecenoic acid (C-16:1 x277;9c) occurred only in the isolates from Trivandrum samples. However, the isolates from Goa did not possess a signature FAME profile. The reproducibility of the GC-MIDI bacterial identification system was evaluated using 16S rRNA gene sequencing techniques for selected isolates.