Bat flies (Diptera: Nycteribiidae and Streblidae) are obligate blood-feeding ectoparasites of bats and are increasingly recognised for their potential role in host-specific co-evolution and disease transmission. Despite their ecological importance, the diversity, host associations and evolutionary relationships of bat flies in Australia remain poorly characterised. This study provides the first integrative assessment of nycteribiid bat flies parasitising Pteropus species in North Queensland, combining morphological taxonomy with cytochrome c oxidase subunit 1 (COX1) sequencing and phylogenetic analysis. A total of 304 bat flies were collected from 79 rescued pteropodids, representing three host species: the little red flying fox (Pteropus scapulatus), the black flying fox (Pteropus alecto) and the spectacled flying fox (Pteropus conspicillatus). Morphological examination of the bat flies identified two taxa, Cyclopodia australis and Cyclopodia albertisii, which were further confirmed by maximum likelihood phylogenetic analysis of COX1 sequences with distinct clade formations delineating species. C. australis was found almost exclusively on little red flying foxes. C. albertisii was mainly associated with both black flying foxes and spectacled flying foxes, with very limited association with the little red flying foxes. These findings underscore the utility of integrative taxonomic approaches in researching bat fly diversity and host specificity. They also highlight the potential for co-evolutionary divergence and emphasise the need for expanded geographic sampling and genomic analysis. This research provides critical baseline data for understanding ectoparasite biodiversity in Australia and contributes to future studies of host–parasite interaction, vector ecology and wildlife disease surveillance.