Aims:  Cyanobacteria‐deprived lichens of the species Canoparmelia caroliniana, Canoparmelia crozalsiana, Canoparmelia texana, Parmotrema sancti‐angeli and Parmotrema tinctorum were screened for the presence of chemo‐organotrophic nitrogen‐fixing bacteria. Methods and Results:  Fifty‐three lichen samples subjected to enrichment selection using a nitrogen‐free minimal medium were positive for acetylene reduction. Seventeen isolates, able to fix nitrogen, belonged to Gamma‐proteobacteria group and were identified as: Acinetobacter sp., Pantoea sp., Pseudomonas sp., Pseudomonas stutzeri, Serratia marcescens and Stenotrophomonas maltophilia, according to 16S rRNA gene sequences and biochemical tests. The excretion of amino acid and phytohormone and the ability of mineral phosphate solubilization were determined in 14 isolates. All isolates were able to release amino acids and 3‐indoleacetic acid. About 64% of the isolates solubilized phosphates and 30% released ethylene. Conclusions:  These data confirm sparse evidence from the literature on the occurrence of chemo‐organotrophic nitrogen‐fixing bacteria in cyanobacteria‐deprived lichens; the isolates presented physiologic features which might benefit the host if they are expressed when the bacteria are harboured by lichens. Significance and Impact of the Study:  Chemo‐organotrophic nitrogen‐fixing bacteria were isolated from a high percentage (72·6%) of cyanobacteria‐deprived lichens. All isolates presented important physiological characteristics, some of which are being described here for the first time.