Aquaculture is the fastest growing food production industry worldwide with production expected to double by 2050 to cope with global protein demand. Barramundi (Lates calcarifer) are an important marine species for Southeast Asian aquaculture. Within these aquaculture industries, disease outbreaks caused by Vibrio harveyi are increasingly being reported. Widely distributed in coastal environments, the prevalence of V. harveyi is predicted to increase as sea-temperatures rise due to climate change. Adding further pressure to Southeast Asian marine aquaculture, climate change is predicted to intensify extreme weather events such as flooding and tropical storms, increasing the physiological stress experienced by fish in these systems. This study investigated the dual effect of cold-stress and V. harveyi infection on the transcriptome (mRNA) and microRNA response of barramundi, 24 hours post-challenge to enhance understanding of host-pathogen interactions. The mRNA response showed cold-stress fish with V. harveyi infection had significant differential gene expression with a total of 11,660 differentially expressed genes compared to 8,744 genes in fish with only V. harveyi infection. All V. harveyi infected fish, regardless of cold-stress, experienced early up-regulation of innate immune factors such as toll-like receptor-13 and hepcidin antimicrobial peptide. However, fish which experienced cold-stress and V. harveyi infection, showed an early pro-inflammatory response with significant up-regulation of interleukin 1 beta, interleukin 6 and tumour necrosis factor alpha. In contrast, the microRNA response was predominantly associated with fish with only V. harveyi infection. Cold-stressed fish with V. harveyi infection had only 8 differentially expressed microRNAs compared to 36 in fish with only V. harveyi infection. MicroRNAs are key regulators of mRNA expression and the lack of regulation observed in fish which experienced cold-stress and V. harveyi could account for the differences in mortality and transcriptome response. These results suggest cold-stress favoured the pathogen, reducing microRNA regulation and resulting in an unregulated pro-inflammatory response. The findings from this study broaden our understanding of the innate immune response of barramundi to V. harveyi and provide insights into microRNA regulation of teleosts in response to bacterial challenge and environmental stressors.