Introduction Tumour necrosis factor inhibitors (anti-TNF agents) are a milestone in rheumatoid arthritis (RA) management, although 20-40% of RA patients do not achieve clinical remission. Identifying reliable biomarkers to predict the therapeutic response to anti-TNF agents remains an unmet clinical need. Methods This study aimed to identify and validate plasma microRNAs (miRNAs) as biomarkers of response to anti-TNF treatment. A two-stage prospective observational (discovery and validation) study was performed. The study population comprised anti-TNF–naïve RA patients and sex- and age-matched healthy controls (HC). Whole miRNA expression was assessed using the Illumina NextSeq 550 platform, followed by bioinformatic analysis. Differentially expressed miRNAs identified in the discovery phase were quantified using real-time quantitative PCR in the validation cohort. Logistic regression analysis assessed associations between miRNA expression and response to anti-TNF treatment. Results A total of 94 participants were included: 70 anti-TNF–naïve RA patients (discovery n = 28; validation n = 42) and 24 healthy controls (14 and 10, respectively). Clinical response at week 24 was defined as DAS28-CRP < 3.2 (responders) versus DAS28-CRP ≥ 3.2 (non-responders). Non-responders were characterised by higher inflammatory activity, poorer HAQ scores, and a higher prevalence of smoking. Forty-five miRNAs were differentially expressed between RA patients and HC, and 9 were expressed between responders and non-responders ( p < 0.05). In the validation cohort, miR-134-5p differed between responders and non-responders (ΔCt Responders = 4.088(±1.266); ΔCt Non-Responders = 5.640(±2.872); ΔΔCt RespondersVsNon-Responders = -1.552; Fold-Change = 2.932; p = 0.024). Logistic regression adjusted for age and sex showed higher miR-134-5p expression to be associated with lack of response to anti-TNF therapy (OR, 1.74; 95% CI: 1.02–2.93; p = 0.039). Conclusion miR-134-5p may serve as a predictive biomarker of poor response to anti-TNF therapy in RA.