Maternally-inherited facultative symbionts are widespread in most insect species, and it is common that several symbionts coexist in the same host individual. Hence, the symbionts may compete or share for the limited resources and space in the host. The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodoidea), harbors a diverse array of facultative symbionts, among which Hamiltonella sp. and Cardinium sp. are abundant species. Hamiltonella alone increases host fitness, while Cardinium alone confers lower fitness. Locking those different partners together creates ideal situations for the evolution of interactions between symbionts. In this study, we compared the fitness effects of whiteflies infected with only Hamiltonella to Hamiltonella-Cardnium co-infected whiteflies and measured the density of Hamiltonella and Cardinium during host aging, aiming to explore Hamiltonella-Cardinium interactions in B. tabaci. Our results illustrated that Hamiltonella-Cardinium coinfection induced lower fecundity, egg hatchability and number of female offspring, leading to a male-biased sex ratio in offspring, while there is no evidence for reproductive incompatibility between the infections. We also found an antagonistic interaction between Hamiltonella and Cardinium given that the density of the latter increased across time and led to a decrease of Hamiltonella density, which may be the underlying causes of the fitness cost in double-infected B. tabaci. Exploring the ecological consequences of co-infections of these different symbionts helps us to understand the nature of host-symbiont interactions in this species and potential for evolutionary conflict.