The gastrointestinal tract commensal microbiome is important for host nutrition, health and immunity. Little information is available regarding the role of these commensals at other mucosal surfaces in poultry. Tracheal mucosal surfaces offer sites for first-line health and immunity promotion in broilers, especially under stress-related conditions. The present study is aimed at elucidating the effects of feed supplementations with mannanoligosaccharides (MOS) prebiotic and a probiotic mixture (PM) on the caecal and tracheal microbiome of broilers kept under chronic heat stress (HS; 35 ± 2°C). Day-old chickens were randomly divided into five treatment groups: thermoneutral control (TN-CONT), HS-CONT, HS-MOS, HS-PM and HS synbiotic (fed MOS and PM). Caecal digesta and tracheal swabs were collected at day 42 and subjected to DNA extraction, followed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. The PCR-DGGE dendrograms revealed significant (49.5% similarity coefficients) differences between caecal and tracheal microbiome. Tracheal microbiome pyrosequencing revealed 9 phyla, 17 classes, 34 orders, 68 families and 125 genera, while 11 phyla, 19 classes, 34 orders, 85 families and 165 genera were identified in caeca. An unweighted UniFrac distance metric revealed a distinct clustering pattern (analysis of similarities, P = 0.007) between caecal and tracheal microbiome. Lactobacillus was the most abundant genus in trachea and caeca and was more abundant in caeca and trachea of HS groups compared with the TN-CONT group. Distinct bacterial clades occupied the caecal and tracheal microbiomes, although some bacterial groups overlapped, demonstrating a core microbiome dominated by Lactobacillus. No positive effects of supplementations were observed on abundance of probiotic bacteria.