BackgroundListeria monocytogenescan survive in cold and wet environments, such as tree fruit packing facilities and it has been implicated in outbreaks and recalls of tree fruit products. However, little is known about microbiota that co-occurs withL. monocytogenesand its stability over seasons in tree fruit packing environments. In this 2-year longitudinal study, we aimed to characterize spatial and seasonal changes in microbiota composition and identify taxa indicative ofL. monocytogenescontamination in wet processing areas of three tree fruit packing facilities (F1, F2, F3).MethodsA total of 189 samples were collected during two apple packing seasons from floors under the washing, drying, and waxing areas. The presence ofL. monocytogeneswas determined using a standard culturing method, and environmental microbiota was characterized using amplicon sequencing. PERMANOVA was used to compare microbiota composition among facilities over two seasons, and abundance-occupancy analysis was used to identify shared and temporal core microbiota. Differential abundance analysis and random forest were applied to detect taxa indicative ofL. monocytogenescontamination. Lastly, threeL. monocytogenes-positive samples were sequenced using shotgun metagenomics with Nanopore MinION, as a proof-of-concept for direct detection ofL. monocytogenes’ DNA in environmental samples.ResultsThe occurrence ofL. monocytogenessignificantly increased from 28% in year 1 to 46% in year 2 in F1, and from 41% in year 1 to 92% in year 2 in F3, while all samples collected from F2 wereL. monocytogenes-positive in both years. Samples collected from three facilities had a significantly different microbiota composition in both years, but the composition of each facility changed over years. A subset of bacterial taxa includingPseudomonas,Stenotrophomonas, andMicrobacterium, and fungal taxa, includingYarrowia,Kurtzmaniella,Cystobasidium,Paraphoma, andCutaneotrichosporon, were identified as potential indicators ofL. monocytogeneswithin the monitored environments. Lastly, the DNA ofL. monocytogeneswas detected through direct Nanopore sequencing of metagenomic DNA extracted from environmental samples.ConclusionsThis study demonstrated that a cross-sectional sampling strategy may not accurately reflect the representative microbiota of food processing facilities. Our findings also suggest that specific microorganisms are indicative ofL. monocytogenes, warranting further investigation of their role in the survival and persistence ofL. monocytogenes.Video AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s40168-023-01544-8.