This dataset contains data from two-part experiments described in the paper: “El Hamss, H., Ghosh, S., M. N., M., Delatte, H., & Colvin, J. (2021). Microbiome diversity and reproductive incompatibility induced by the prevalent endosymbiont Arsenophonus in two species of African cassava Bemisia tabaci whiteflies. Ecology and Evolution, 00, 1–10. https://doi.org/10.1002/ece3.840”. The experiment investigates the effects of Arsenophonus on whitefly reproduction and microbiome diversity. In the first experiment (“crossing experiment”), the effect of Arsenophonus is studied on number of eggs, nymphs, males, and females on different whitefly species at intraspecies level (SSA1-SG3A+ with SSA1-SG3A-) and at interspecies level (SSA2A+ with SSA1-SG3A- and SSA1-SG3A+). Arsenophonus infection A+ means the presence of Arsenophonus in related whitefly colony, A- means the absence of Arsenophonus. In the second experiment (“microbiome diversity”), whitefly colonies of crossed parents and generated progenies were sequenced to check Arsenophonus threshold (number of reads) and the presence of other bacteria in those crossed whiteflies. The main results of this work are: (i) Arsenophonus did not induce reproductive incompatibility within SSA1-SG3 but reduced the number of eggs, nymphs and female ratios, (ii) complete RI was observed between SSA1-SG3 and SSA2 indicating the lack of gene flow between the two whitefly species, (iii) many new ‘other bacteria’ in SSA B. tabaci have been identified, whose role remains to be investigated.