Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be an important mechanism of virulence. While S. pneumoniae employs a multitude of glycan processing enzymes, the exo-mannosidase SpGH92 has been shown to be an important virulence factor. Accordingly, SpGH92 is hypothesized to be a target for much-needed new treatments of S. pneumoniae infection. Here we report the synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (Manα1,2Manβ-4MU) as a fluorogenic disaccharide substrate and development of an assay for SpGH92 that overcomes its requirement for +1 binding site occupancy. We miniaturize our in vitro assay and apply it to a high-throughput screen of >65 000 compounds, identifying a single inhibitory chemotype, LIPS-343. We further show that Manα1,2Manβ-4MU is also a substrate of the human Golgi-localized α-mannosidase MAN1A1, suggesting that this substrate should be useful for assessing the activity of this and other mammalian α-mannosidases.