Karst aquifers are highly vulnerable to pollution from human activities. Among sources of these contaminants, septic tank effluent can easily pollute karst aquifers, especially concentrated inputs such as those, for example, from tourist hotels. However, the impacts of septic effluent from relatively large, concentrated inputs on karst aquifers have seldom been assessed previously and therefore provide the focus of this study. Artificial tracer tests, geochemical analysis, and dual nitrate stable isotopes were employed to evaluate the impacts of a concentrated input of septic effluent from the Jinfoshan Holiday Hotel (JHH) on the vulnerable Shuifang Spring (SFS) karst aquifer in a remote mountainous area, the Jinfoshan Karst World Heritage Site within Chongqing Municipality of southwest China. The results of artificial tracer tests showed that the underground flow mainly occurred in a primary conduit with a pooled or bifurcated flow path that connects a sinkhole input to SFS. The high tracer recovery rates suggest that the karst aquifer was characterized by high intrinsic vulnerability to contamination. Chemographs at SFS responded rapidly to the episodic release of effluent from JHH. Decreased pH and dissolved oxygen and elevated turbidity, specific conductance and NH concentrations of SFS resulted from the episodic release of septic tank effluent from the JHH during high-use periods. Although the nitrate concentrations were far below the guideline value of the Standard for Groundwater Quality of China, the isotopes of δN and δO suggest that nitrate flowing from SFS was primarily derived from manure and sewage, in addition to soil organic N. Thus, episodic release of septic effluent provides a challenge to the sustainability of karst groundwater management. The results of this study may be relevant to other remote and mountainous karst environments where tourism provide otherwise scarce economic resources and particularly to protected sites throughout the world.