In cooperation with the New York City Department of Environmental Protection (NYC DEP), the U.S. Geological Survey (USGS) collected water and bed sediment samples along Alley Creek (Queens, New York) to help determine likely sources of fecal bacteria to the creek and Little Neck Bay. Potential terrestrial sources include stormwater, sewage via combined sewer overflow (CSO) and compromised infrastructure, bed-sediment resuspension, and groundwater discharge. Host sources that were targeted using microbial source tracking (MST) techniques included human, canine, waterfowl, and general Bacteroides. Routine water samples were collected and analyzed for the fecal indicator bacteria enterococci and fecal coliform, along with the physicochemical constituent total suspended solids (TSS), at the NYC DEP Newton Creek Microbiological Laboratory (Brooklyn, New York) and for MST markers at the USGS Ohio Water Microbiological Laboratory (OWML; Columbus, Ohio)--these results from routine samples are available at the USGS National Water Information System (NWIS) Web interface (https://waterdata.usgs.gov/nwis/qw). Data in this data release were generated from the two local investigations of NYC DEP sites TI-008 and TI-24 collected to supplement routine monitoring. At TI-008, sediment and water samples were collected along a stormwater conveyance line between Oakland Lake and Alley Creek on September 22, 2020 to conduct a sediment resuspension experiment. In May 2021, a total of 20 surface water and depth-profile samples (taken from a hole in a 7-foot submerged wastewater pipe adjacent to TI-024), along with thermal imagery, were collected adjacent to combined sewer overflow outfall TI-024.