Southern Portugal is experiencing a rapid change in land use due to the spread of intensive farming systems, namely olive production systems, which can cause strong negative environmental impacts and affect the ecological integrity of aquatic ecosystems. This study aimed to identify the main environmental disturbances related with olive grove intensification on Mediterranean-climate streams in southern Portugal, and to evaluate their effects on fish assemblage structure and integrity. Twenty-six stream sites within the direct influence of traditional, intensive, and hyper-intensive olive groves were sampled. Human-induced disturbances were analyzed along the olive grove intensity gradient. The integrity of fish assemblages was evaluated by comparison with an independent set of least disturbed reference sites, considering metrics and guilds, based on multivariate analyses. Along the gradient of olive grove intensification, the study observed overall increases in human disturbance variables and physicochemical parameters, especially organic/nutrient enrichment, sediment load, and riparian degradation. Animal load measured the impact of livestock production. This variable showed an opposite pattern, since traditional olive groves are often combined with high livestock production and are used as grazing pasture by the cattle, unlike more intensive olive groves. Stream sites influenced by olive groves were dominated by non-native and tolerant fish species, while reference sites presented higher fish richness, density and were mainly occupied by native and intolerant species. Fish assemblage structure in olive grove sites was significantly different from the reference set, although significant differences between olive grove types were not observed. Bray-Curtis similarities between olive grove sites and references showed a decreasing trend in fish assemblage integrity along the olive grove intensification gradient. Olive production, even in traditional groves, led to multiple in-stream disturbances, whose cumulative effects promoted the loss of biota integrity. The impacts of low intensity traditional olive groves on aquatic ecosystems can be much greater when they are coupled with livestock production. This paper recommends best practices to reduce negative impacts of olive production on streams, contributing to guide policy decision-makers in agricultural and water management.