While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2,603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all 15 hits impaired global protein synthesis and upregulated CDKN1A (p21) levels, but caused diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We defined RPS28 as a direct target of the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, which caused a severe pre-18S pre-rRNA processing defect, and uncovered SPRR3's novel role in promoting RNAP1 transcription. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics.