Mycobacterium canettii
is a smooth bacillus related to the
Mycobacterium tuberculosis
complex. It causes lymph nodes and pulmonary tuberculosis in patients living in countries of the Horn of Africa, including Djibouti. The environmental reservoirs of
M
.
canettii
are still unknown. We aimed to further decrypt these potential reservoirs by using an original approach of High-Throughput Carbon and Azote Substrate Profiling. The Biolog Phenotype profiling was performed on six clinical strains of
M
.
canettii
and one
M
.
tuberculosis
strain was used as a positive control. The experiments were duplicated and authenticated by negative controls. While
M
.
tuberculosis
metabolized 22/190 (11%) carbon substrates and 3/95 (3%) nitrogen substrates, 17/190 (8.9%) carbon substrates and three nitrogen substrates were metabolized by the six
M
.
canettii
strains forming the so-called corebiologome. A total at 16 carbon substrates and three nitrogen substrates were metabolized in common by
M
.
tuberculosis
and the six
M
.
canettii strains
. Moreover, at least one
M
.
canettii
strain metabolized 36/190 (19%) carbon substrates and 3/95 (3%) nitrogen substrates for a total of 39/285 (13%) substrates. Classifying these carbon and nitrogen substrates into ten potential environmental sources (plants, fruits and vegetables, bacteria, algae, fungi, nematodes, mollusks, mammals, insects and inanimate environment) significantly associated carbon and nitrogen substrates metabolized by at least one
M
.
canettii
strain with plants (
p
= 0.006). These results suggest that some plants endemic in the Horn of Africa may serve as ecological niches for
M
.
canettii
. Further ethnobotanical studies will indicate plant usages by local populations, then guiding field microbiological investigations in order to prove the definite environmental reservoirs of this opportunistic tuberculous pathogen.