AbstractThe floral nectar of angiosperms harbors a variety of microorganisms that depend predominantly on animal visitors for their dispersal. Although some members of the genusAcinetobacterand all currently known species ofRosenbergiellaare thought to be adapted to thrive in nectar, there is limited information about the response of these bacteria to variation in the chemical characteristics of floral nectar. We investigated the growth performance of a diverse collection ofAcinetobacter(n= 43) andRosenbergiella(n= 45) isolates obtained from floral nectar and the digestive tract of flower-visiting bees in a set of 12 artificial nectars differing in sugar content (15% w/v or 50% w/v), nitrogen content (3.48/1.67 ppm or 348/167 ppm of total nitrogen/amino nitrogen), and sugar composition (only sucrose, 1/3 sucrose + 1/3 glucose + 1/3 fructose, or 1/2 glucose + 1/2 fructose). Growth was only observed in four of the 12 artificial nectars. Those containing elevated sugar concentration (50% w/v) and low nitrogen content (3.48/1.67 ppm) were limiting for bacterial growth. Furthermore, phylogenetic analyses revealed that the ability of the bacteria to grow in different types of nectar is highly conserved between closely related isolates and genotypes, but this conservatism rapidly vanishes deeper in phylogeny. Overall, these results demonstrate that the ability ofAcinetobacterspp. andRosenbergiellaspp. to grow in floral nectar largely depends on nectar chemistry and bacterial phylogeny.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00248-022-02088-4.