The Large-footed Myotis Myotis macropus is a threatened echolocating bat that uses a specialised ‘trawling' foraging strategy to hunt for aquatic prey. While the species is well known in freshwater habitats, in 2014 it was recorded for the first time roosting and foraging in a sheltered bay on Sydney Harbour in the Port Jackson estuary. To investigate how widely distributed M. macropus was within the estuary (Parramatta River, Lane Cove River, Middle Harbour, harbour islands, west Harbour and east Harbour), 56 sampling sites were surveyed acoustically. Of these sites, 24 were in harbour bays/coves, 20 were in tributary bays, seven were along tributary channels/creeks, four were on the margins of harbour islands and a single site was located on a freshwater lake. We also investigated relationships between M. macropus activity and environmental variables to identify those that should be targeted for management. Radio-tracking of M. macropus at one known roost was carried out to assess roost fidelity and identify key foraging areas within the estuary. Myotis macropus was widespread in Port Jackson, being present at 92.6 % of sites, but with high activity (>90 passes night−1), including feeding buzzes (≥24.5 buzzes night−1) concentrated in a few ‘hot spots’. Greatest activity was recorded in east Harbour (~70 passes night−1), west Harbour (~15.5) and Lane Cove River (~14), while lowest activity was on the Parramatta River (2) and Middle Harbour (10). Activity, including feeding, was significantly greater in harbour bays/coves when compared with other habitats. Radio-tracking revealed that bats roosting in west Harbour showed 100 % fidelity to the roost site over a three week period and were only recorded foraging in this zone and the nearby (1.2 km) Lane Cove River. Historical Zinc concentrations in surficial sediments was negatively associated with M. macropus activity, though heavy metals were also correlated positively with total suspended solids (TSS). While heavy metal concentrations in sediments were not associated with feeding activity, increased TSS was negatively associated with M. macropus activity and feeding. Best subsets regressions found that M. macropus activity was associated with TSS (−ve), mangrove cover (+ve), seagrass cover (+ve) and total water extent (−ve). We recommend further research on the negative association between TSS, heavy metals and M. macropus activity to identify the sensitivity of this species to past and present pollution events.