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Twelve Azotobacter isolates exhibited varying levels of biofilm formation in a microtiter plate assay. The most efficient biofilm-forming isolate, as identified by partial 16S rRNA gene sequencing analysis, was Azotobacter vinelandii (AZCH6). Two isolates (AZCH5 and AZCH6), showing similar plant growth-promoting traits but differing in in vitro biofilm forming capacity, were further investigated for biofilm characterization on glass coverslips and on roots of wheat seedlings using confocal laser scanning microscopy and scanning electron microscopy. Rhizosphere colonization by AZCH6 in terms of CFU g−1 of soil, was significantly higher (P≤0.05) as compared to AZCH5. The data revealed that AZCH6 and AZCH5 exhibits similar pattern between biofilm formation and rhizosphere colonization. Therefore, strong biofilm-forming ability of Azotobacter on plant roots should be considered an important criterion for selection of effective isolates for enhanced root colonization.